Vibrational spectroscopy of bacteriorhodopsin mutants: I. Tyrosine‐185 protonates and deprotonantes during the photocycle

Mark S. Braiman, Tatsushi Mogi, Lawrence J. Stern, Neil R. Hackett, Betty H. Chao, H. Gobind Khorana, Kenneth J. Rothschild

Research output: Contribution to journalArticlepeer-review

89 Scopus citations

Abstract

The techniques of FTIR difference spectroscopy and site‐directed mutagenesis have been combined to investigate the role of individual tyrosine side chains in the proton‐pumping mechanism of bacteriorhodopsin (bR). For each of the 11 possible bR mutants containing a single Tyr→Phe substitution, difference spectra have been obtained for the bR→K and bR→M photoreactions. Only the Tyr‐185→Phe mutation results in the disappearance of a set of bands that were previously shown to be due to protonation of a tryosinate during the br→K photoreaction [Rothschild et al.: Proceedings of the National Academy of Sciences of the United states of America 83:347, (1986)]. The Tyr‐185→Phe mutation also eliminates a set of bands in the bR→M difference spectrum associated with deprotonation of a Tyr; most of these bands (e.g., positive 1272‐cm−1 peak) are completely unaffected by the other ten Tyr→Phe mutations. Thus, tyrosinate‐185 gains a proton during the bR→K reaction and loses it again when M is formed. Our FTIR spectra also provide evidence that Tyr‐185 interacts with the protonated Schiff base linkage of the retinal chromophore, since the negative CNH+ stretch band shifts from 1640 cm−1 in the wild type to 1636 cm−1 in the Tyr‐185→Phe mutant. A model that is consistent with these results is that Tyr‐185 is normally ionized and serves as a counter‐ion to the protonated Schiff base. The primary photoisomerization of the chromophore translocates the Schiff base away from Tyr‐185, which raises the pKa of the latter group and results in its protonation.

Original languageEnglish (US)
Pages (from-to)219-229
Number of pages11
JournalProteins: Structure, Function, and Bioinformatics
Volume3
Issue number4
DOIs
StatePublished - 1988

Keywords

  • Schiff base counter‐ion
  • energy transduction
  • proton transport
  • proton wire
  • purple membrane

ASJC Scopus subject areas

  • Structural Biology
  • Biochemistry
  • Molecular Biology

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